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Patologias hereditárias raras associadas à sobrecarga em ferro
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Abstract(s)
O ferro é um nutriente essencial para diversos processos biológicos. No entanto, tanto o seu défice como o seu excesso no organismo têm consequências negativas para a saúde. A Hemocromatose Hereditária (HH) é um conjunto de doenças genéticas que resultam de uma absorção excessiva e desregulada deste metal proveniente da alimentação e da sua deposição em órgãos como fígado, coração, pâncreas e glândulas endócrinas. Os sintomas mais frequentes desta doença podem ir deste fatiga e artralgia até cardiomiopatias, cirrose ou carcinoma hepatocelular. O gene HFE que codifica para o regulador homeostático do ferro, quando mutado, é o principal gene associado à patologia na sua forma comum, de desenvolvimento tardio (HH clássica ou tipo I). Contudo, existem outros genes relacionados com o metabolismo do ferro que poderão estar envolvidos no desenvolvimento de outro tipo de HH, mais rara e com características particulares (HH não-clássica). Entre estes, encontram-se os genes HAMP e HJV que codificam, respectivamente, para a hepcidina e hemojuvelina, e que poderão estar na origem de HH com características clínicas mais graves e de início precoce (HH tipo II ou HH juvenil). Por outro lado, o gene TFR2, que codifica para o receptor 2 da transferrina, poderá estar associado a HH tipo III e o gene da ferroportina (SLC40A1) relacionado com a HH tipo IV ou com a Doença da Ferroportina, ambas com transmissão autossómica dominante.
Um dos objectivos deste trabalho consistiu na implementação, no laboratório, de uma nova metodologia de análise rápida, de baixo custo e em larga escala de pesquisa de alterações em seis genes relacionados com o metabolismo do ferro (HFE, HAMP, HJV, TFR2, SLC40A1 e FTL), através de PCR-longos e posterior sequenciação por next-generation sequencing, recorrendo à metodologia Nextera XT (Illumina). Foram então analisados, por esta metodologia, 30 indivíduos com fenótipos sugestivos de HH não clássica. Após análise bioinformática dos resultados obtidos, foram identificadas 89 variantes genéticas diferentes para as quais se realizaram estudos in silico. Recorreu-se à ferramenta bioinformática PolyPhen-2, de modo a analisar o impacto das variantes missense ao nível da estrutura e função das respectivas proteínas. Por outro lado, pesquisou-se o possível efeito das variantes genéticas ao nível do splicing, utilizando o software Human Splicing Finder. As variantes consideradas patogénicas ou possivelmente patogénicas foram confirmadas por sequenciação de Sanger. Assim, foi possível, identificar várias alterações genéticas raras, algumas delas observadas pela primeira vez na população portuguesa, como por exemplo, a SLC40A1:c.610G>A ou a TFR2:c.2136+708A>G e compreender o respectivo mecanismo molecular subjacente ao desenvolvimento da respectiva patologia.
Neste estudo também se pretendeu pesquisar o possível efeito modulador de uma variante comum no gene BMP2 (SNP rs235768) e de variantes no promotor do HAMP sobre o desenvolvimento de sobrecarga em ferro numa população que já apresentava um factor genético de susceptibilidade para o desenvolvimento de hiperferritinémia (p.His63Asp no gene HFE). A população analisada (n=138) já tinha sido previamente genotipada para as variantes no promotor do gene HAMP, pelo que foi agora genotipada para a alteração c.570A>T no gene BMP2 por PCR-ARMS. A análise estatística realizada com recurso ao software SPSS revelou que nenhuma destas variantes está a contribuir para o fenótipo de sobrecarga em ferro, na população analisada.
Concluímos que, com este estudo, contribuímos para o conhecimento da base molecular da HH não-clássica na população portuguesa, esclarecemos a relação genótipo/fenótipo nalguns dos casos complexos estudados e contribuímos para aumentar o conhecimento sobre a fisiopatologia e os mecanismos subjacentes ao desenvolvimento deste tipo de patologias complexas relacionadas com a desregulação do metabolismo do ferro.
Iron is an essential nutrient for several biological processes. However, both its deficit and excess in the body have negative health consequences. Hereditary Hemochromatosis (HH) is a set of genetic diseases that result from excessive and unregulated absorption of this metal from food and its deposition in organs such as liver, heart, pancreas and endocrine glands. The most common symptoms of this disease may range from fatigue and arthralgia to cardiomyopathies, cirrhosis or hepatocellular carcinoma. The HFE gene coding for homeostatic iron regulator, when mutated, is the main gene associated with pathology in its common, late-developing form (classic or type I HH). However, there are other genes related to iron metabolism that may be involved in the development of another type of HH, rarer and with particular characteristics (non-classical HH). These include the HAMP and HJV genes coding for hepcidin and hemojuvelin, respectively, which may be the origin of HH with more severe and early-onset clinical features (juvenile or type II HH). On the other hand, the transferrin receptor 2 encoded by the TFR2 gene may be associated with type III HH and type IV HH-related ferroportin gene or Ferroportin Disease, both with autosomal dominant transmission. One of the objectives of this work was the implementation in the laboratory of a new method for rapid, low cost and large scale analysis of changes in six genes related to iron metabolism (HFE, HAMP, HJV, TFR2, SLC40A1 and FTL), by long-range PCR and subsequent sequencing by next-generation sequencing, using the Nextera XT methodology (Illumina). Thirty individuals with phenotypes suggestive of non-classical HH were then analyzed. After bioinformatics analysis of the results obtained, 89 different genetic variants were identified for which in silico studies were performed. PolyPhen-2 software was used to analyze the impact of missense variants on the structure and function of their proteins. The possible effect of splicing genetic variants was investigated using the Human Splicing Finder software. Pathogenic or possibly pathogenic variants were confirmed by Sanger sequencing. Thus, it was possible to identify several rare genetic changes, some of them first observed in the Portuguese population, such as SLC40A1:c.610G>A or TFR2:c.2136+708A>G and to understand their molecular mechanism underlying the development of the respective pathology. This study also intended to investigate the possible modulating effect of a common variant on the BMP2 gene (SNP rs235768) and HAMP promoter variants on the development of iron overload in a population that already had a genetic susceptibility factor for the development of hyperferritinemia. (p.His63Asp in HFE). The population analyzed (n=138) had previously been genotyped for HAMP promoter variants and was now genotyped for the c.570A> T change in the BMP2 gene by PCR-ARMS. Statistical analysis using the SPSS software revealed that none of these variants is contributing to the iron overload phenotype in the population analyzed. In conclusion, with this study we have contributed to the knowledge of the molecular basis of the non-classical HH in the Portuguese population and clarified the genotype/phenotype relationship in some of the uncommon cases studied. Furthermore, we contributed to the understanding of the pathophysiology and mechanisms underlying the development of these complex pathologies related to iron metabolism deregulation.
Iron is an essential nutrient for several biological processes. However, both its deficit and excess in the body have negative health consequences. Hereditary Hemochromatosis (HH) is a set of genetic diseases that result from excessive and unregulated absorption of this metal from food and its deposition in organs such as liver, heart, pancreas and endocrine glands. The most common symptoms of this disease may range from fatigue and arthralgia to cardiomyopathies, cirrhosis or hepatocellular carcinoma. The HFE gene coding for homeostatic iron regulator, when mutated, is the main gene associated with pathology in its common, late-developing form (classic or type I HH). However, there are other genes related to iron metabolism that may be involved in the development of another type of HH, rarer and with particular characteristics (non-classical HH). These include the HAMP and HJV genes coding for hepcidin and hemojuvelin, respectively, which may be the origin of HH with more severe and early-onset clinical features (juvenile or type II HH). On the other hand, the transferrin receptor 2 encoded by the TFR2 gene may be associated with type III HH and type IV HH-related ferroportin gene or Ferroportin Disease, both with autosomal dominant transmission. One of the objectives of this work was the implementation in the laboratory of a new method for rapid, low cost and large scale analysis of changes in six genes related to iron metabolism (HFE, HAMP, HJV, TFR2, SLC40A1 and FTL), by long-range PCR and subsequent sequencing by next-generation sequencing, using the Nextera XT methodology (Illumina). Thirty individuals with phenotypes suggestive of non-classical HH were then analyzed. After bioinformatics analysis of the results obtained, 89 different genetic variants were identified for which in silico studies were performed. PolyPhen-2 software was used to analyze the impact of missense variants on the structure and function of their proteins. The possible effect of splicing genetic variants was investigated using the Human Splicing Finder software. Pathogenic or possibly pathogenic variants were confirmed by Sanger sequencing. Thus, it was possible to identify several rare genetic changes, some of them first observed in the Portuguese population, such as SLC40A1:c.610G>A or TFR2:c.2136+708A>G and to understand their molecular mechanism underlying the development of the respective pathology. This study also intended to investigate the possible modulating effect of a common variant on the BMP2 gene (SNP rs235768) and HAMP promoter variants on the development of iron overload in a population that already had a genetic susceptibility factor for the development of hyperferritinemia. (p.His63Asp in HFE). The population analyzed (n=138) had previously been genotyped for HAMP promoter variants and was now genotyped for the c.570A> T change in the BMP2 gene by PCR-ARMS. Statistical analysis using the SPSS software revealed that none of these variants is contributing to the iron overload phenotype in the population analyzed. In conclusion, with this study we have contributed to the knowledge of the molecular basis of the non-classical HH in the Portuguese population and clarified the genotype/phenotype relationship in some of the uncommon cases studied. Furthermore, we contributed to the understanding of the pathophysiology and mechanisms underlying the development of these complex pathologies related to iron metabolism deregulation.
Description
Dissertação de Mestrado em Biologia Humana e Ambiente, apresentado à Faculdade de Ciências da Universidade de Lisboa, 2019
Co-orientadora Paula Faustino, Departamento de Genética Humana do Instituto Nacional de Saúde Doutor Ricardo Jorge.
Co-orientadora Paula Faustino, Departamento de Genética Humana do Instituto Nacional de Saúde Doutor Ricardo Jorge.
Keywords
Metabolismo do Ferro Doenças Raras Hemocromatose Hereditária NGS Doença da Ferroportina Modificadores Genéticos Doenças Genéticas Genética Humana Iron Metabolism Iron Overload Hereditary Hemochromatosis Genetic Variants